Transovarial transmission of Yersinia pestis in its flea vector, Xenopsylla cheopis

Yersinia pestis is the causative agent of bubonic plague, a deadly flea-borne disease responsible for three historic pandemics. Today annual cases of human disease occur worldwide following exposure to Y. pestis infected fleas that can be found within the rodent population where plague activity cycles between epizootic outbreaks and extended periods of apparent quiescence. Flea transmission of Y. pestis is most efficient in “blocked” fleas that are unable to feed, whereas mammalian transmission to fleas requires a susceptible host with end-stage high titer bacteremia. These facts suggest alternative mechanisms of transmission must exist to support the persistence of Y. pestis between epizootic outbreaks. In this work, we addressed whether vertical transmission could be a mechanism for persistent low-infection across generations of fleas. We demonstrate that Y. pestis infection of the Oriental rat flea, Xenopyslla cheopis, spreads to the reproductive tissues and is found in eggs produced by infected adult fleas. We further show that vertical transmission of Y. pestis from eggs to adults results in midgut colonization indicating a strong probability that it can reenter the sylvatic plague cycle.


Introduction
Yersinia pestis is the causative agent of plague, which is classi ed as a prioritized re-emerging zoonotic disease within the United States.Worldwide plague foci persist due to enzootic sylvatic cycles consisting of burrowing rodents and their respective ea vectors, which are a crucial component for transmission 9- 15 .Numerous ea species and primarily rodent hosts are implicated in maintaining the sylvatic plague cycle, with mice, rats, prairie dogs, and ground squirrels commonly found in established plague foci 16,17 .Fleas can transmit Y. pestis as early as day 1 post-infection, in a poorly understood mechanism involving regurgitation from the midgut with an enhanced e cacy that results from high ea density 18 .Once in the midgut, bacteria respond to environmental cues that activate expression and production of an exopolysaccharide matrix, which develops into an infectious bio lm 19,20 .During feeding the bio lm localized to the proventriculus occludes ingestion and causes regurgitation which deposits Y. pestis in the dermis of the human or animal host.From this site, as few as one bacterium is su cient to cause lethal bubonic plague.
Early studies of the rodent-ea plague cycle documented proventricular blockage and its potential correlation to transmission in eas known to vector Y. pestis.Blockage and transmission by blocked eas was most frequent in the rat ea, Xenopsylla cheopis, with more than 50% occurrence, whereas blockage was very infrequent in other plague vectors including Oropsylla montana 21 .Recent work demonstrated that occurrence of proventricular blockage in Y. pestis-infected O. montana could be enhanced by using rat blood feeding compared to mouse blood 22 .This observation suggests that increased populations of rats should increase the likelihood of epizootic plague, however this is not observed in naturally occurring plague foci.In addition to bio lm-mediated proventricular blockage, Y. pestis can be transmitted through a regurgitative mechanism shortly after the ea becomes infected 23 .This mechanism appears to be independent of blood meal source, but can also result in non-productive transmission suggesting it may slow epizootic spread of disease 24 .Although these two methods of transmission are believed to require distinct proteins from the bacteria and eas, both occur from the midgut.There have been no reports of Y. pestis localization to salivary glands, and no indication that bacteria are able to escape the midgut of eas.Thus, the collective data led to the prevailing model for Y. pestis transmission involving continuous cycling between adult eas and susceptible rodent hosts.With these parameters, the persistence of Y. pestis in the environment where there is little to no plague activity cannot be modeled.
In this work, we therefore addressed the historic paradigm that Y. pestis is con ned to the midgut and alimentary canal in eas with the underlying hypothesis that vertical transmission in the ea could account for long periods of reduced plague activity in a given endemic area.Using a highly sensitive uorescent reporter with confocal microscopy and additional imaging with transmission electron microscopy, we reexamined the distribution of Y. pestis following membrane feeding of X. cheopis in male and female eas and their progeny.

Results
Yersinia pestis escapes to reproductive tissue in the ea vector Xenopsylla cheopis.
To understand Y. pestis interactions in eas, we used a highly sensitive expression system for the uorescent reporter tdTomato as a means to localize bacteria following infection of X. cheopis eas in an arti cial membrane feeder.With this approach, we established the behavior of uorescent Y. pestis in the midgut using confocal microscopy and followed an apparent Y. pestis-containing biomass that developed and peaked in the midgut on day 3 post-infection (Fig. 1A-D).In some of the eas collected mainly on day 3, brightly uorescing Y. pestis could be seen in the esophagus, similar to previous reports (representative shown in Fig. 1C) 22,25 .By day 7, the Y. pestis containing biomass decreased, consistent with previous studies (Fig. 1D-E).The kinetics of the biomass suggests correlation with competency for early phase transmission.In parallel, bacterial load was quanti ed in pools of 3 midguts per group, demonstrating similar median values for colony forming units (CFU) across the 7-day time course (Fig. 1F).These data establish that while the kinetics of the Y. pestis-containing biomass change during the 7-day course of infection, viable bacterial load is relatively constant in the ea midgut.
To determine whether Y. pestis may be localized outside of the midgut, we examined the ovarioles and the testes of eas dissected from the various times post-infection.Using confocal microscopy, we generated z-stacks to visualize the inside of the ovarioles and testes.Indeed, Y. pestis was readily observable, appearing to localize inside the ovarioles rather than on the outside surface (Fig. 2A-D).Similarly, we readily observed tomato-uorescing Y. pestis in the testes of male adult eas (Fig. 2E-H).Therefore, perhaps not unexpectedly, Y. pestis was also found in the spermatheca (Fig. 2I-L).Other tissues that contained Y. pestis that was visible by confocal microscopy included Malpighian tubules, but not salivary glands (Extended Data Fig. 1, salivary glands not shown).Furthermore, Y. pestis could be seen in the reproductive organs up to 15 weeks after the adult eas were infected (Extended Data Fig. 2).This result suggests that Y. pestis infection of the reproductive tissue is stable throughout the lifespan of the adult ea.
Viable Y. pestis is present in the oviposited eggs and larvae derived from infected adult eas.
To determine whether viable Y. pestis was present in the eggs, we devised an egg collection chamber, using sifting to separate the eggs from bedding debris.Adult eas that are feeding regularly have a steady reproduction at 3.6 to 5.6 eggs per female per day which decreases as the eas age 26,27 .Eggs from X. cheopis are 477-504 µm long with a width of 297-333 µm and off-white in color.Sifting was carried out every 1-3 days and eggs were collected of the expected size and color.Eggs were thoroughly washed and then processed for imaging or for plating to determine bacterial titer.Control eas, given blood that was not infected, produced eggs with no uorescent signal either on or within the egg tissue (Fig. 3A-D).In sharp contrast, tdTomato uorescence was abundant in the interior of eggs produced by Y. pestis-infected eas, localizing to nearly the length of the egg (Fig. 3E-L).We found prominent uorescent signal in more than half of the eggs that were tested, whether they were collected 3 (Fig. 3D-F) or 7 (Fig. 3I-L) days postinfection.Considering blood intake initiates copulation and subsequent oviposition, the data suggest that egg-derived Y. pestis were either transferred directly to the egg due to midgut escape during the bloodmeal, or that colonization of the spermatheca results in the continuous opportunity for Y. pestis to infect newly developing eggs 28 .
We also collected larvae by sifting and found 5 of 8 were positive, with tdTomato uorescence that appeared to be present in nearly the entire length of the larvae (Fig. 4D-I) compared to larvae from control eas which were uninfected (Fig. 4A-C).To rule out the likelihood that larvae were infected by eating Y. pestis that had been expelled from the hindgut, we collected eggs that were deposited by Y. pestis-infected adult X. cheopis and reared them in a separate sterile container into larvae.As expected, uorescent Y. pestis was readily visualized in these samples (Fig. 4G-I).Together, these data indicate that Y. pestis in the eggs survives development into the larval stage.Additional eggs from Y. pestis-infected adult eas were collected, placed into sterile sawdust with sterile larvae food where they developed into pupae and F1 adults.Pupae exhibited a high degree of auto uorescence (Extended Data Fig. 3A-B).Nevertheless, strong tdTomato expression could be detected in the pupae reared from Y. pestis-infected eggs (Extended Data Fig. 3C-D).Likewise, uorescent Y. pestis could also be visualized in the midguts of the F1 adult eas (Extended Data Fig. 4A-D).Bacteria harvested from these eas were screened for Y. pestis plasmidencoded genes, caf1 and pla by PCR, and indeed, the transovarially-transmitted bacteria were positive for both genes, suggesting they retained the extrachromosomal plasmids (Extended Data Fig. 4E).
Transovarially acquired Y. pestis is competent for transmission.
To determine whether transovarially transmitted bacteria were viable, we compared bacterial titers in eggs, larvae, pupae and F1 progeny.The median number of colonies recovered from eggs was only 5 CFU (Fig. 5A).In the later stages of development, bacteria appeared to replicate, with median titer recovered in larvae of approximately 50 CFU, and in pupae, the median titer was over 100 CFU.In the newly emerged adult F1 eas collected prior to their rst blood feeding, two midguts were harvested and combined for plating, resulting in 173 CFU recovered.To con rm the retention of extrachromosomal plasmids, bacterial stocks were made and then used for PCR to amplify the Y. pestis plasmid-speci c genes caf1 and pla.Following PCR, the expected-size bands were puri ed and processed to determine the DNA sequence in order to conclusively con rm Y. pestis (Extended Data Fig. 4E, sequence data not shown).Together, these data indicate that transovarially transmitted Y. pestis remain viable throughout the developmental cycle and the recovery of viable Y. pestis from the midgut of F1 adult eas suggests that transovarially transmitted bacteria are competent for transmission.
We therefore evaluated whether Y. pestis isolated from eggs remained competent for ea transmission to a mammalian host.Naïve adult eas were challenged with egg-isolated Y. pestis in the arti cial membrane feeder and on days 3 and 7, infected eas were used in a transmission study.On day 3 post-infection, it was evident that egg-isolated Y. pestis had been successfully transmitted, and its localization to midgut biomass appeared indistinguishable from eas infected with the original Y. pestis stock (Fig. 5B-C).Similar kinetics of egg-isolated Y. pestis were observed in the ea midgut as seen for the parent Y. pestis strain with similar bacterial load observed on day 7 post-infection.Overall, these data suggest that transovarially transmitted Y. pestis is competent for re-entry into the sylvatic plague cycle.
Transmission electron microscopy (TEM) shows Y. pestis localized to reproductive tissue in adult eas.
To con rm that bacteria were present in the testes and ovaries of infected adult eas, we used transmission electron microscopy to image tissues that were dissected from male and female eas.In the females, we observed bacteria in the ovaries, and even in the safety pin morphology that is characteristic of Y. pestis (Fig. 6B-C).These bacteria were not found in eas that were not infected (Fig. 6A).In the male eas, we readily identi ed abundant Y. pestis in the basal membrane of the testes, appearing on the distal side of the spermatocyte follicle, often within membrane-enclosed vesicles (Fig. 6D-F).This suggests that Y. pestis may be intracellular in the reproductive tissue.Overall, the TEM con rms that Y. pestis exit the midgut and localize to the reproductive organs of an adult ea.

Discussion
In this work, we have provided multiple layers of evidence that transovarial transmission occurs in X. cheopis infected with Y. pestis.These ndings provide visual, quantitative, and genetic support that low levels of Y. pestis escape from the midgut throughout the lifespan of an infected ea, entering the reproductive tract where they are able to colonize progeny eggs.In the more than 100 years of Y. pestis research, escape from the alimentary tract has never been reported.Previous studies were heavily focused on salivary glands, and in agreement with these results, we did not observe any Y. pestis in the salivary glands.Furthermore, the amount of colony forming units harvested from ea eggs is exceedingly small, averaging fewer than 10 CFU, consequently it may simply be that previous experiments were not sensitive enough to capture the low level of transovarial transmission.In this work, the high-level expression of the tdTomato reporter revealed a new understanding of sylvatic plague.
Fleas are vectors of other diseases, namely rickettsiosis and cat scratch fever caused by Rickettsia felis or typhi or Bartonella henselae, respectively.For Rickettsia typhi, transovarial transmission was observed in eas more than 30 years ago 29 .Like Rickettsia felis, intracellular Y. pestis appear to localize to the ovariole tissue 30 .However, there are likely signi cant differences in the underlying mechanism of midgut escape.Whereas R. felis infection of the midgut epithelium was readily apparent shortly after infection, there was no detectable infection of these cells by Y. pestis.Following invasion of the midgut epithelium R. felis was observed in hemocoel prior to its systemic dissemination to the reproductive tract and salivary glands.In contrast, Y. pestis was not observed in hemocoel or salivary glands.
Transovarial transmission could be a contributor to maintenance of Y. pestis within the environment.From the data shown here, viable Y. pestis is continuously deposited in eggs, suggesting most, if not all, progeny eas remain infected.Furthermore, the strong selective pressure of the sylvatic plague cycle would favor the retention of virulence factors by transovarially-transmitted Y. pestis.Therefore, it seems likely that transovarial transmission could sustain low levels of Y. pestis in a rodent community.Environmental factors such as precipitation and temperature are predicted to have an impact on the transovarial transmission cycle, thereby having multi-layered contributions to the prevalence of Y. pestis in the environment.By understanding transovarial transmission, we may improve our ability to model the enzootic cycle of plague.Competing interests: The authors declare no competing interests.
Additional information: Supplementary information is available for this paper.

Data Availability:
The sequence chromatographs and alignments, image data sets and corresponding metadata that support the ndings of this study are available in Figshare with the identi er 10.6084/m9.gshare.c.6845496.[37]   All unique biological materials, including bacterial strains and plasmids, are available from the corresponding author upon request.There are no restrictions on any of these materials.
Materials and Correspondence: Correspondence and requests for materials should be addressed to Dr. Deborah Anderson, andersondeb@missouri.edu.
Reprints and permissions information is available at www.nature.com/reprints.work did not reintroduce the pCD1 plasmid, and therefore are classi ed as select agent-exempt strains by the US Center for Disease Control and Prevention.Prior to use in infection, laboratory reared, naive X. cheopis were separated from the colony and starved for at least ve days and no more than seven days to improve feeding e ciency during infection.Groups of 50 eas were infected with Y. pestis strain KIM6+ carrying plasmid-expression of the uorescent protein tdTomato (Excitation: 554, Emission 581) 34 .An arti cial membrane feeder was constructed using skin from an adult mouse.Blood was inoculated with 5x10 8 to 1x10 9 Y. pestis and maintained at 37°C, and eas were allowed to feed for 1 hour.The species of animal blood (rat, mouse, pig, or prairie dog) used in the arti cial feeder is indicated in the gure legends.
When eas were removed from the feeder, they were observed to determine intake of the bloodmeal.Fleas that had not fed were removed from the study.
Midgut processing: Fleas were euthanized on days 1, 3 and 7 post-infection without additional blood feeding.For experiments that lasted more than 7 days, eas were provided maintenance blood meal every 7 days throughout the duration.Dissected midguts and other reproductive tissues were isolated and placed onto sterile slides, xed with 4% paraformaldehyde, and rinsed 3 times with PBS for a minimum of 30 each time to mounting.
Confocal Midguts, eggs, larvae and pupae were imaged using a SP8 confocal microscope.quanti cation of midgut biomass, images were converted to 8-bit grayscale for quanti cation of observed integrated density (ID O ).Control eas (n=10), fed in parallel with uninfected blood and analyzed 1 day after feeding, were used to determined background uorescence.Background signal and midgut area were used to normalize the samples and calculate integrated density (ID= ID O -(midgut area x background)).Image J software was used to and quantify the signal intensity, reported as relative uorescent units (RFU) 35 .
Quanti cation of load: For plating, individual samples were homogenized in 10μL sterile PBS, midguts were pooled in groups of 3; serial dilutions were performed in sterile PBS and all dilutions were plated in duplicate onto heart infusion agar (HIA) or Yersinia selective agar (YSA).Isolated colonies from eggs, larvae, pupae, and F1 adults were streaked for isolation before storing in bacteriological freezing media at -80°C.collection: Infected eas using rat blood were maintained in modi ed housing with 300-micron mesh on lid, 550-micron mesh under the bedding, a lower chamber that be easily removed.Eggs and larvae were collected by sifting, care avoid with infected feces eas.Fleas were maintained in the modi ed housing and provided uninfected rat bloodmeal every 7 days.Sifting for eggs and larvae occurred every days post-infection.To aseptically collect the eggs and larvae, a cotton was moistened double-distilled, sterile H 2 O and used to up eggs or larvae.These specimens were placed onto a sterile slide, washed sterile PBS three times, observed between washing to ensure no materials or feces were in contact, then mounted in 35% glycerol for confocal microscopy or transferred to sterile PBS.After washing three times in sterile PBS, a new sterile, moistened cotton applicator was used to transfer the egg to an agar plate.Each egg was punctured with a sterile needle, releasing the contents onto the agar.Similarly, larvae were washed three times with sterile PBS, then homogenized and plated.Development of Y. pestis-infected eggs or larvae: Eggs were isolated, washed in sterile PBS, and placed into a sterile ea chamber with mesh on the top and bottom containing sterile sawdust and larval food.These samples were imaged or plated on agar after development into larvae, pupae, or F1 adults.
PCR and genome annotation: Bacteria that were isolated from eggs were grown overnight and DNA was isolated using a Quick-DNA® Microprep kit (Zymo Research, California, USA).Conventional PCR ampli cation of caf1 and pla were performed using the primers shown in Extended Data Table 1.All positive PCRs were con rmed by sequencing; sequenced nucleotides were aligned in Geneious Prime using MAFFT 36 .
Transmission study: Transmission assays were carried out as previously described for eas infected with KIM6+ptdTomato 8 .For assays using bacteria originally harvested from eggs, minor modi cations were made.Brie y, egg isolated bacteria were used to infect adult eas on an arti cial feeder.On day 3 or 7 post-infection, groups of 10 eas were used for the transmission assay and fed on uninfected rat bloodmeal in the arti cial membrane feeder for 1 hour.Blood and skin were processed to quantify Y. pestis by plating to determine the number of transmitted per Transmission electron microscopy.Following the same methods, eas were either fed an uninfected rat bloodmeal or a Y. pestis-infected rat bloodmeal and groups of 10 were separated based on sex.On day 3, eas were euthanized, and the ovaries, testes, and midguts were dissected, xed in 2% paraformaldehyde, 2% glutaraldehyde in 100 mM sodium cacodylate buffer with a pH of 7.35.Each sample was allowed to settle, the resulting tissue was resuspended in Histogel (ThermoScienti c, Kalamazoo, MI).Tissues were rinsed in 100 mM sodium cacodylate buffer with a pH of 7.35 containing 130 mM sucrose.Secondary xation performed using osmium tetroxide (Ted Pella, Inc. Redding, California) in cacodylate buffer.Specimens were incubated at 4°C for then rinsed with cacodylate buffer and further with distilled water.En staining was performed using 1% aqueous uranyl acetate and at 4°C then rinsed with distilled water.A graded dehydration series was performed using ethanol, transitioned into acetone, and dehydrated tissues were in ltrated with Epon and polymerized at 60°C overnight.Sections cut to thickness of 75 nm using ultramicrotome UCT, Leica Microsystems, Germany) and a diamond knife (Diatome, Hat eld PA).Images were acquired with a JEOL JEM 1400 transmission microscope (JEOL, Peabody, MA) at 80 kV on a Rio CMOS (Gatan, Inc, Pleasanton, CA).All samples were prepared, processed and imaged at the University of Electron Microscopy Statistical analysis.Data was grouped based on trial and microscopic analysis of individual ea midguts and tissue, with controls groups from the parent Y. pestis and uninfected eas.Descriptive statistics were evaluated using SPSS and graphed using OriginPro.Transstadially transmitted Yersinia pestis retains plasmids and capability for transmission (A) Eggs and larvae were collected from Y. pestis -infected X. cheopis eas and either processed or reared to pupae and F1 adult eas.Samples were processed to quantify bacterial load; F1 adult midguts were pooled (n=2), n=7 eggs, n=2 larvae, n=4 pupae.(B-C) Y. pestis isolated from eggs collected from infected adults was used to infect naïve X. cheopis eas.On days 3 and 7 post-infection, a subset of infected eas was used

Figures
Declarations of Missouri Genomics Technology Core; microscopy was conducted at the University of Missouri Advanced Light Microscopy Core; and transmission electron microscopy was conducted at the Electron Microscopy Core at the University of Missouri.This work was partially supported by PHS NIH/NIAID 1R21AI178547 and the University of Missouri System Tier 2 Strategic Investment Program.Author contributions: C.P. designed and performed experiments, interpreted data and co-wrote the manuscript; B.B. and Q.S. contributed to data interpretation and editing of the manuscript; D.A. contributed to experimental design and interpretation, and co-wrote the manuscript.All authors approved the manuscript.